Glucose Transport In L6 Myotubes Research Articles

Current trends in small molecule discovery targeting key cellular signaling events towards the combined management of diabetes and obesity.

Non-insulin dependent diabetes mellitus, also known as Type 2 diabetes is a polygenic disorder leading to abnormalities in the carbohydrate and lipid metabolism. The major contributors in the pathophysiology of type 2 diabetes (T2D) include resistance to insulin action, β cell dysfunction, an abnormality in glucose metabolism and storage, visceral obesity and to some extent inflammation and oxidative stress. Insulin resistance, along with a defect in insulin secretion by the pancreatic β cells is instrumental towards progression to hyperglycemia. Increased incidence of obesity is also a major contributing factor in the escalating rates of type 2 diabetes. Drug discovery efforts are therefore crucially dependent on identifying individual molecular targets and validating their relevance to human disease. The current review discusses bioactive compounds from medicinal plants offering enhanced therapeutic potential for the combined patho-physiology of diabetes and obesity.We have demonstrated that 3β-taraxerol a pentacyclic triterpenoid (14-taraxeren-3-ol) isolated from the ethyl acetate extract of Mangifera indica, chlorogenic acid isolated from the methanol extract of Cichorium intybus, methyl tetracosanoate from the methanol extract of Costus pictus and vitalboside A derived from methanolic extract of Syzygium cumini exhibited significant effects on insulin stimulated glucose uptake causing insulin sensitizing effects on 3T3L1 adipocytes (an in vitro model mimicking adipocytes). Whereas, (3β)-stigmast-5-en-3-ol isolated from Adathoda vasica and Aloe emodin isolated from Cassia fistula showed significant insulin mimetic effects favoring glucose uptake in L6 myotubes (an in vitro model mimicking skeletal muscle cells). These extracts and molecules showed glucose uptake through activation of PI3K, an important insulin signaling intermediate. Interestingly, cinnamic acid isolated from the hydro-alcohol extract of Cinnamomum cassia was found to activate glucose transport in L6 myotubes through the involvement of GLUT4 via the PI3K-independent pathway. However, the activation of glucose storage was effective in the presence of 3β-taraxerol and aloe emodin though inhibition of GSK3β activity. Therefore, the mechanism of improvement of glucose and lipid metabolism exhibited by the small molecules isolated from our lab is discussed. However, Obesity is a major risk factor for type-2 diabetes leading to destruction of insulin receptors causing insulin resistance. Identification of compounds with dual activity (anti-diabetic and antiadipogenic activity) is of current interest. The protein tyrosine phosphatase 1B (PTP1B) is an important negative regulator of the insulin and leptin-signaling pathway is of significance in target definition and discovery.

Ketoisocaproic acid, a metabolite of leucine, suppresses insulin-stimulated glucose transport in skeletal muscle cells in a BCAT2-dependent manner.

Although leucine has many positive effects on metabolism in multiple tissues, elevated levels of this amino acid and the other branched-chain amino acids (BCAAs) and their metabolites are implicated in obesity and insulin resistance. While some controversies exist about the direct effect of leucine on insulin action in skeletal muscle, little is known about the direct effect of BCAA metabolites. Here, we first showed that the inhibitory effect of leucine on insulin-stimulated glucose transport in L6 myotubes was dampened when other amino acids were present, due in part to a 140% stimulation of basal glucose transport (P < 0.05). Importantly, we also showed that α-ketoisocaproic acid (KIC), an obligatory metabolite of leucine, stimulated mTORC1 signaling but suppressed insulin-stimulated glucose transport (-34%, P < 0.05) in an mTORC1-dependent manner. The effect of KIC on insulin-stimulated glucose transport was abrogated in cells depleted of branched-chain aminotransferase 2 (BCAT2), the enzyme that catalyzes the reversible transamination of KIC to leucine. We conclude that although KIC can modulate muscle glucose metabolism, this effect is likely a result of its transamination back to leucine. Therefore, limiting the availability of leucine, rather than those of its metabolites, to skeletal muscle may be more critical in the management of insulin resistance and its sequelae.

Deoxyandrographolide promotes glucose uptake through glucose transporter-4 translocation to plasma membrane in L6 myotubes and exerts antihyperglycemic effect in vivo

Skeletal muscle is the principal site for postprandial glucose utilization and augmenting the rate of glucose utilization in this tissue may help to control hyperglycemia associated with diabetes mellitus. Here, we explored the effect of Deoxyandrographolide (DeoAn) isolated from the Andrographis paniculata Nees on glucose utilization in skeletal muscle and investigated its antihyperglycemic effect in vivo in streptozotocin-induced diabetic rats and genetically diabetic db/db mice. In L6 myotubes, DeoAn dose-dependently stimulated glucose uptake by enhancing the translocation of glucose transporter 4 (GLUT4) to cell surface, without affecting the total cellular GLUT4 and GLUT1 content. These effects of DeoAn were additive to insulin. Further analysis revealed that DeoAn activated PI-3-K- and AMPK-dependent signaling pathways, account for the augmented glucose transport in L6 myotubes. Furthermore, DeoAn lowered postprandial blood glucose levels in streptozotocin-induced diabetic rats and also suppressed the rises in the fasting blood glucose, serum insulin, triglycerides and LDL-Cholesterol levels of db/db mice. These findings suggest the therapeutic efficacy of the DeoAn for type 2 diabetes mellitus and can be potential phytochemical for its management.

Enhancement of glucose uptake by Ficus hispida methanolic extract through phosphatidylinositol 3 kinase and inhibiting aldose reductase – An in vitro analysis

IntroductionType 2 diabetes mellitus is a polygenic metabolic syndrome characterized by impaired carbohydrate and lipid metabolism. Diabetes mellitus is commonly associated with secondary disorders such as obesity and genetic predisposition. Prolonged hyperglycemia can also lead to secondary complications such as cataractogenesis, retinopathy and nephropathy through the induction of the polyol pathway. Ficus hispida (FH) is a medium size tree found in India, Sri Lanka and the Andaman Islands in damp localities. Traditionally, different parts of the FH plant have been used in the treatment of various diseases and disorders. MethodologyFH bark was sequentially extracted using different organic solvents ranging from non polar to polar. Glucose uptake assays were performed using 2-deoxy-d-1-[3H] for the FH extracts. Fluorimetric analysis was adopted to assess the aldose reductase inhibitory effect. The gene and protein level expression of the markers were analyzed using RT-PCR and Western blot analysis. ResultsF. hispida methanolic extract (FHME) showed an enhancement in the glucose uptake and upregulated the gene and protein level expression of IRβ, IRS-1, PI3K, GLUT4 and PPARγ. Comparison with wortmannin, a specific PI3K inhibitor confirmed that the active FHME recruits glucose uptake through a PI3K dependent pathway. Subsequently, FHME possesses a significant aldose reductase inhibitory activity. ConclusionThe current study emphasizes the cellular level mechanism of FHME on glucose transport in L6 myotubes and the inhibition of aldose reductase activity.

Riluzole increases the rate of glucose transport in L6 myotubes and NSC-34 motor neuron-like cells via AMPK pathway activation

Riluzole is the only approved ALS drug. Riluzole influences several cellular pathways, but its exact mechanism of action remains unclear. Our goal was to study the drug's influence on the glucose transport rate in two ALS relevant cell types, neurons and myotubes. Stably transfected wild-type or mutant G93A human SOD1 NSC-34 motor neuron-like cells and rat L6 myotubes were exposed to riluzole. The rate of glucose uptake, translocation of glucose transporters to the cell's plasma membrane and the main glucose transport regulatory proteins’ phosphorylation levels were measured. We found that riluzole increases the glucose transport rate and up-regulates the translocation of glucose transporters to plasma membrane in both types of cells. Riluzole leads to AMPK phosphorylation and to the phosphorylation of its downstream target, AS-160. In conclusion, increasing the glucose transport rate in ALS affected cells might be one of the mechanisms of riluzole's therapeutic effect. These findings can be used to rationally design and synthesize novel anti-ALS drugs that modulate glucose transport in neurons and skeletal muscles.

Regulation of GLUT1‐mediated glucose and dehydroascorbic acid (DHA) transport

The GLUT1 PDZ‐binding motif that plays a role in GLUT1 localization contains a reported phosphorylation site, S490, for ataxia telangiectasia mutated (ATM). The objective of this study is to determine whether ATM and/or modification of GLUT1 S490 affect GLUT1 localization, trafficking, and transport activity. Mouse extensor digitorum longus (EDL) muscles were excised from wild type and ATM −/− mice and then subjected to a DHA transport assay. ATM inhibition by KU55933 or CP466722 or ATM dysfunction (ATM−/−) in EDL resulted in reduced DHA transport (~30–65%, p&lt;0.05). Also, L6 myoblasts were transfected with FLAG tagged GLUT1 (FLAG‐GLUT1) or FLAG‐GLUT1 S490A and S490D site mutants. ATM inhibition significantly diminished cell surface FLAG‐GLUT1 (~20–80%, p&lt;0.05) and glucose transport in L6 myotubes (~50%, p&lt;0.05) with a concomitant reduction in GLUT1 externalization (~20%, p&lt;0.05). The S490D mutation increased cell surface GLUT1 (~35%, p&lt;0.05) as well as GLUT1‐mediated glucose transport (~2‐fold, p&lt;0.05), while S490A had opposite effects. These results provide evidence that ATM and GLUT1 S490 mutations affect cell surface GLUT1 and GLUT1‐mediated transport. Supported by USPHS award 1R15DK091904.

Mechanisms for skeletal muscle insulin resistance in patients with pancreatic ductal adenocarcinoma

Objective Weight loss, glucose intolerance, and insulin resistance are seen in patients with pancreatic ductal adenocarcinoma (PDAC). Peripheral insulin resistance is decreased after tumor resection in patients with PDAC, which is consistent with the hypothesis that factors from the tumor may induce skeletal muscle insulin resistance. Our aim was to investigate the possible mechanisms for their skeletal muscle insulin resistance. Accordingly, the action of insulin on glucose metabolism and content of energy metabolites in muscle of patients with PDAC were investigated. To explore whether PDAC cells could influence muscle glucose uptake, myotubes were exposed to media conditioned by PDAC cells. Methods Muscle biopsies from patients with PDAC ( n = 13), cancer of other sites ( n = 8), chronic pancreatitis ( n = 8), and controls with benign diseases ( n = 8) were assessed for glycogen, adenosine triphosphate, and phosphocreatine content. Basal and insulin-stimulated glucose transport and incorporation into glycogen were also assessed. Myotubes were treated with media conditioned by PDAC (MiaPaca 2) cells and glucose transport was monitored. Results Insulin-stimulated glucose transport, muscle glycogen, and adenosine triphosphate content were decreased in patients with PDAC compared with controls, and insulin stimulation did not significantly increase glucose incorporation into glycogen in vitro in patients with PDAC. Adenosine triphosphate content correlated with glycogen content but not with glucose transport in skeletal muscle. Media conditioned with human PDAC cells did not affect basal or insulin-stimulated glucose transport in L6 myotubes. Conclusion In patients with PDAC, muscle insulin resistance is an early and specific finding unrelated to weight loss, plasma free fatty acid levels, and energy status of the cell. PDAC cell-derived factors did not directly induce insulin resistance in myotubes, suggesting a lack of direct tumor-related effects.

Anti-diabetic Activities of Ethanol Extracts from Persimmon Leaves

Diospyros kaki L. (persimmon), cultivated in Korea for several centuries, is thought to have originated in southern China. The persimmon leaves are approved as “Traditional Chinese Medicine” in China for the treatment of stroke and apoplexy syndrome [Fan et al., 2006], and are utilized as a hypotensive drug in Japan [Funayama et al., 1979]. Furthermore, in Korea the leaves have long been used as herb tea and were traditionally employed for the treatment of hypertension in patients with type 2 diabetes mellitus [Sa et al., 2005]. The condensed tannins and flavonoids from the persimmon leaves were found to be mainly responsible for the antihypertensive, antimutagenic, anticarcinogenic, and antioxidative actions [Choo et al., 2000]. However, little studies have been performed on the anti-diabetic properties of the persimmon leaves. Type 2 diabetes mellitus is a heterogeneous metabolic disorder characterized by the impaired insulin secretion from the pancreatic beta cells and the insulin resistance in the peripheral tissues such as liver, adipose tissue, and skeletal muscle [Ward et al., 1984]. Skeletal muscles account for approximately 75% of the glucose absorption under the insulin-stimulated conditions, and glucose transport is the rate-limiting step in the primary glucose disposal and utilization, especially in the insulin-targeted skeletal muscle. Thus, the alteration of the glucose transport is one of the major defects in type 2 diabetes both in vitro and in vivo [Dohm et al., 1988; Ziel et al., 1988; DeFronzo et al., 1992]. For these reasons, some targeted anti-diabetic drugs have been developed; for example, metformin (dimethylbiguanide), a commonly used glucose-lowering reagent, is thought to increase the glucose uptake in the skeletal muscle. [Musi et al., 2002]. Rosiglitazone and pioglitazone both act by reducing the glucose production in the liver and increasing the insulindependent glucose uptake in the muscle cells [Yonemitsu et al., 2001]. Therefore, we investigated the anti-diabetic therapeutic potential of the ethanol extracts of the persimmon leaves to maximize the glucose transport in L6 myotubes, which have been widely used to study the mechanism of the insulin-stimulated glucose transport. Persimmon leaves (0.5 kg) were purchased from the herbal medicine co-operative association in Daejeon, Korea. The dried leaves were extracted with ethanol (1 L ×2), filtered,and evaporated. The ethanol extracts were then suspended in DMSO for further experiments. L6 rat myoblasts were obtained from the American Type Culture Collection, cultured in DMEM containing 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin, and incubated at 37oC under 5% CO2 humidified atmosphere. For the differentiation of myoblasts into myotubes, the medium was switched to DMEM with 2% horse serum for 6 days. To assess the anti-diabetic effects of the ethanol extracts of persimmon leaves, we conducted glucose transport assays at various concentrations (20, 40, 60, and 80 μg/ mL) (Fig. 1); incubation of the myotubes with the ethanol extracts of persimmon leaves for 1 h significantly enhanced the insulin-stimulated glucose transport in a dose-dependent manner by 114.3, 123, 160.7, and 183.7%, respectively, as compared to the negative control with the insulin treatment. These results suggest that persimmon leaves affect the insulin-stimulated muscular glucose transport, and, therefore, could be used as insulin sensitizers for the treatment of diabetes. To investigate the role of the ethanol extracts of persimmon leaves on the insulin receptor-regulated glucose transport activity, we assessed the tyrosine phosphatase1B in vitro. As a critical negative regulator of the insulin signal transduction cascade, tyrosine phosphatase1B acts to reverse the tyrosine kinase action and is a key phosphatase for the insulin receptor and the insulin receptor substrate-1, major mediators of the glucose transport pathways [Moller et al., 2001]. Tyrosine phosphatase1B has also been implicated in the insulin-dependent pathways, as well as in the insulin insensitivity that is the most common pathology of type 2 diabetes and obesity [Na et al., 2006]. Therefore, the *Corresponding author Phone: +82-42-821-6834; Fax: +82-42-821-8887 E-mail: cbsohn@cnu.ac.kr

Selective cyclooxygenase-2 inhibitors stimulate glucose transport in L6 myotubes in a protein kinase Cδ-dependent manner

Selective inhibitors of cyclooxygenase-2 (prostaglandin-endoperoxide synthase-2; COX-2) augment the rate of hexose uptake in myotubes by recruiting glucose transporter-4 (GLUT-4) to the plasma membrane in an insulin- and AMPKα-independent manner [Alpert E, Gruzman A, Lardi-Studler B, Cohen G, Reich R, Sasson S. Cyclooxygenase-2 (PTGS2) inhibitors augment the rate of hexose transport in L6 myotubes in an insulin- and AMPKα-independent manner. Diabetologia 2006;49:562–70]. We aimed at elucidating the molecular interactions that mediate this effect of COX-2 inhibitors in L6 myotubes. The effects of the inhibitors niflumic acid, nimesulide and rofecoxib on activities and phosphorylation state of key proteins in the insulin transduction pathway were determined. These inhibitors did not induce specific tyrosine phosphorylation in IRS-1, could not assemble a functional IRS-PI3K-PKB/Akt complex and did not activate GSK3α/β, JNK1/2, ERK1/2, p38-MAPK or c-Cbl by site-specific phosphorylation(s). Yet, like insulin, they activated mTOR and induced downstream threonine phosphorylation in p70S6K and 4EBP1. However, rapamycin, which inhibits mTOR enzymatic activity, did not interfere with COX-2 inhibitor-induced stimulation of hexose uptake in myotube. Thus, mTOR activation was not required for COX-2 inhibitor-dependent augmentation of hexose transport in myotubes. Because PKCδ has also been shown to activate mTOR, we asked whether COX-2 inhibitors activate mTOR by a prior activation of PKCδ. Indeed, all three inhibitors induced tyrosine phosphorylation in PKCδ and stimulated its kinase activity. Moreover, pharmacological inhibition of PKCδ or the expression of a dominant-negative form of PKCδ in myotubes completely abolished COX-2 inhibitor-dependent stimulation of hexose uptake. This study shows that selective COX-2 inhibitors activate a unique PKCδ-dependent pathway to increase GLUT-4 abundance in the plasma membrane of myotubes and augment the rate of hexose transport.

Protein Kinase C-Independent Effects of Protein Kinase D3 in Glucose Transport in L6 Myotubes

Protein kinase C (PKC) and protein kinase D (PKD) coordinate and regulate many fundamental cellular processes. In this study, we evaluate the role of classic and novel PKC (c/nPKC) and PKD in glucose transport in L6 myotubes. c/nPKC is either activated by short-term phorbol 12-myristate 13-acetate (PMA) treatment or down-regulated by prolonged PMA treatment at a high dose in L6 myotubes. Our results indicate that PMA treatments have little impact on basal and insulin-stimulated glucose uptake and insulin-induced Akt activation. In contrast, the PKC inhibitors Go6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo[3,4-c] carbazole], Go6983 [2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl)maleimide], GF 109203X [bisindolylmaleimide I; 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(1H-indol-3-yl)maleimide], and Ro 31-8220 [bisindolylmaleimide IX; 2-{1-[3-(amidinothio)propyl]-1H-indol3-yl}-3-(1-methylindol-3-yl)maleimide] block basal and insulin-stimulated glucose uptake, and their inhibitory effects persist upon down-regulation of c/nPKC by PMA, implying the presence of PKC-independent effectors in mediating their inhibition of glucose uptake. Go6976, the potent cPKC inhibitor that also effectively inhibits PKD, dose-dependently blocks basal glucose uptake in L6 myotubes, whereas Go6983, the nonselective PKC inhibitor that is ineffective for PKD, has little effect on basal glucose uptake, implying the involvement of PKD in this process. Most prominently, adenoviral gene expression of a dominant-negative PKD isoform, PKD3, primarily inhibits basal glucose uptake and, to a lesser extent, insulin-stimulated glucose uptake, whereas overexpression of wild-type PKD3 significantly enhances basal glucose uptake. Moreover, expression of a PKD3-targeted siRNA significantly inhibits basal glucose uptake. Taken together, our results indicate that PKD, specifically PKD3, directly contributes to insulin-independent basal glucose uptake in L6 skeletal muscle cells.

Troglitazone induces GLUT4 translocation in L6 myotubes.

A number of studies have demonstrated that insulin resistance in the skeletal muscle plays a pivotal role in the insulin resistance associated with obesity and type 2 diabetes. A decrease in GLUT4 translocation from the intracellular pool to the plasma membranes in skeletal muscles has been implicated as a possible cause of insulin resistance. Herein, we examined the effects of an insulin-sensitizing drug, troglitazone (TGZ), on glucose uptake and the translocation of GLUT4 in L6 myotubes. The prolonged exposure (24 h) of L6 myotubes to TGZ (10(-5) mol/l) caused a substantial increase in the 2-deoxy-[3H]D-glucose (2-DG) uptake without changing the total amount of the glucose transporters GLUT4, GLUT1, and GLUT3. The TGZ-induced 2-DG uptake was completely abolished by cytochalasin-B (10 micromol/l). The ability of TGZ to translocate GLUT4 from light microsomes to the crude plasma membranes was greater than that of insulin. Both cycloheximide treatment (3.5 x 10(-6) mol/l) and the removal of TGZ by washing reversed the 2-DG uptake to the basal level. Moreover, insulin did not enhance the TGZ-induced 2-DG uptake additively. The TGZ-induced 2-DG uptake was only partially reversed by wortmannin to 80%, and TGZ did not change the expression and the phosphorylation of protein kinase B; the expression of protein kinase C (PKC)-lambda, PKC-beta2, and PKC-zeta; or 5'AMP-activated protein kinase activity. a-Tocopherol, which has a molecular structure similar to that of TGZ, did not increase 2-DG uptake. We conclude that the glucose transport in L6 myotubes exposed to TGZ for 24 h is the result of an increased translocation of GLUT4. The present results imply that the effects of troglitazone on GLUT4 translocation may include a new mechanism for improving glucose transport in skeletal muscle.

Effects of adenoviral gene transfer of wild-type, constitutively active, and kinase-defective protein kinase C-lambda on insulin-stimulated glucose transport in L6 myotubes.

We used adenoviral gene transfer methods to evaluate the role of atypical protein kinase Cs (PKCs) during insulin stimulation of glucose transport in L6 myotubes. Expression of wild-type PKC-lambda potentiated maximal and half-maximal effects of insulin on 2-deoxyglucose uptake, but did not alter basal uptake. Expression of constitutively active PKC-lambda enhanced basal 2-deoxyglucose uptake to virtually the same extent as that observed during insulin treatment. In contrast, expression of kinase-defective PKC-lambda completely blocked insulin-stimulated, but not basal, 2-deoxyglucose uptake. Similar to alterations in glucose transport, constitutively active PKC-lambda mimicked, and kinase-defective PKC-lambda completely inhibited, insulin effects on GLUT4 glucose transporter translocation to the plasma membrane. Expression of kinase-defective PKC-lambda, in addition to inhibition of atypical PKC enzyme activity, was attended by paradoxical increases in GLUT4 and GLUT1 glucose transporter levels and insulin-stimulated protein kinase B enzyme activity. Our findings suggest that in L6 myotubes, 1) atypical PKCs are required and sufficient for insulin-stimulated GLUT4 translocation and glucose transport; and 2) activation of protein kinase B in the absence of activation of atypical PKCs is insufficient for insulin-induced activation of glucose transport.

Effects of Adenoviral Gene Transfer of Wild-Type, Constitutively Active, and Kinase-Defective Protein Kinase C- on Insulin-Stimulated Glucose Transport in L6 Myotubes

Effects of Adenoviral Gene Transfer of Wild-Type, Constitutively Active, and Kinase-Defective Protein Kinase C- on Insulin-Stimulated Glucose Transport in L6 Myotubes

Evidence for involvement of protein kinase C (PKC)-zeta and noninvolvement of diacylglycerol-sensitive PKCs in insulin-stimulated glucose transport in L6 myotubes.

We examined the question of whether insulin activates protein kinase C (PKC)-zeta in L6 myotubes, and the dependence of this activation on phosphatidylinositol (PI) 3-kinase. We also evaluated a number of issues that are relevant to the question of whether diacylglycerol (DAG)-dependent PKCs or DAG-insensitive PKCs, such as PKC-zeta, are more likely to play a role in insulin-stimulated glucose transport in L6 myotubes and other insulin-sensitive cell types. We found that insulin increased the enzyme activity of immunoprecipitable PKC-zeta in L6 myotubes, and this effect was blocked by PI 3-kinase inhibitors, wortmannin and LY294002; this suggested that PKC-zeta operates downstream of PI 3-kinase during insulin action. We also found that treatment of L6 myotubes with 5 microM tetradecanoyl phorbol-13-acetate (TPA) for 24 h led to 80-100% losses of all DAG-dependent PKCs (alpha, beta1, beta2, delta, epsilon) and TPA-stimulated glucose transport (2-deoxyglucose uptake); in contrast, there was full retention of PKC-zeta, as well as insulin-stimulated glucose transport and translocation of GLUT4 and GLUT1 to the plasma membrane. Unlike what has been reported in BC3H-1 myocytes, TPA treatment did not elicit increases in PKCbeta2 messenger RNA or protein in L6 myotubes, and selective retention of this PKC isoform could not explain the retention of insulin effects on glucose transport after prolonged TPA treatment. Of further interest, TPA acutely activated membrane-associated PI 3-kinase in L6 myotubes, and acute effects of TPA on glucose transport were inhibited, not only by the PKC inhibitor, LY379196, but also by both wortmannin and LY294002; this suggested that DAG-sensitive PKCs activate glucose transport through cross-talk with phosphatidylinositol (PI) 3-kinase, rather than directly through PKC. Also, the cell-permeable, myristoylated PKC-zeta pseudosubstrate inhibited insulin-stimulated glucose transport both in non-down-regulated and PKC-depleted (TPA-treated) L6 myotubes; thus, the PKC-zeta pseudosubstrate appeared to inhibit a protein kinase that is required for insulin-stimulated glucose transport but is distinct from DAG-sensitive PKCs. In keeping with the latter dissociation of DAG-sensitive PKCs and insulin-stimulated glucose transport, LY379196, which inhibits PKC-beta (preferentially) and other DAG-sensitive PKCs at relatively low concentrations, inhibited insulin-stimulated glucose transport only at much higher concentrations, not only in L6 myotubes, but also in rat adipocytes, BC3H-1 myocytes, 3T3/L1 adipocytes and rat soleus muscles. Finally, stable and transient expression of a kinase-inactive PKC-zeta inhibited basal and insulin-stimulated glucose transport in L6 myotubes. Collectively, our findings suggest that, whereas PKC-zeta is a reasonable candidate to participate in insulin stimulation of glucose transport, DAG-sensitive PKCs are unlikely participants.

Evidence for Involvement of Protein Kinase C (PKC)- and Noninvolvement of Diacylglycerol-Sensitive PKCs in Insulin-Stimulated Glucose Transport in L6 Myotubes

Evidence for Involvement of Protein Kinase C (PKC)- and Noninvolvement of Diacylglycerol-Sensitive PKCs in Insulin-Stimulated Glucose Transport in L6 Myotubes